In order to determine the concentration of analytes in biological fluids, specific binding partners for analytes are often used. The analyte concentration is determined by generating a signal which is modulated in accordance with the amount of analyte bound to the binding partner. Many forms of binding assays have been described, most of which depend on a physical technique such as centrifugation or filtration to separate bound from free material. These techniques can be complex and expensive to automate. On the other hand, the optical phenomenon known as "total internal reflection" has been used to distinguish bound from free material in several immunoassay systems that do not require a mechanical separation step. Immunoassay systems employ antibodies as the analyte binding partners; the principles used, however, can be applied to other forms of binding assays such as those using hormone receptors or DNA probes.
Total internal reflection is a phenomenon that occurs when light is aimed at a glancing angle, above the so-called "critical angle", from a medium of high refractive index, such as glass, toward a medium of lower refractive index, such as water. The beam of light is reflected at the interface between the two media. Total internal reflection is described in E. Hecht and A. Zajac, Optics, Addison-Wesley Publishing Co., Reading, Mass. (1974), pp. 81-84.
Under conditions of total internal reflection, it can be demonstrated that a portion of the light called the "evanescent wave" penetrates the low-refractive-index medium to a depth of a fraction of a wavelength, typically 100 nm or so. This light will therefore illuminate materials which are bound at the interface between the two media; materials not at the interface will not be illuminated. This provides a separation, a means of distinguishing bound from free material, without the need for a mechanical separation device.
U.S. Pat. No. 3,939,350 (Kronick et al.) describes an immunoassay system employing haptens or antibodies attached to a glass prism having a surface in contact with an aqueous medium. Immunologically-bound fluorescent antibodies are detected by their presence within the region illuminated by the evanescent wave. To achieve this, Kronick designed a system such that light enters the sample chamber above the critical angle. This requires sophisticated light sources such as lasers or arc lamps to produce a small diameter, collimated beam for illumination of a small sample. The samples are placed on a slide but nevertheless the sample chamber must be cleaned after each assay.
U.S. Pat. No. 4,451,434 (Hart) utilizes fluorescent latex particles as a label, giving potentially much greater signal per binding event than that obtainable by Kronick et al. Although an improvement over Kronick et al., Hart still is faced with the problem of using sophisticated light sources with the inherent disadvantages just related. Also, high quality sample cuvettes must be used which are formed to incorporate prisms. Even when well manufactured, plastic devices will not in general have the high optical quality of the glass, quartz or sapphire prisms.
EP 0 326 375 and EP 0 254 430 (Schutt et al.) describe a similar immunoassay system in which light-scattering particles, such as polymer latex or colloidal gold, are used in place of the fluorophores described by Kronick et al. The examples in these patents indicate that assays employing evanescent wave phenomena can achieve sensitivity otherwise obtainable only in assays that employ a mechanical separation step. Otherwise, Schutt et al. suffer from the same disadvantages as Hart and Kronick et al.
U.S. Pat. No. 4,447,546 (Hirschfeld) is an example of the use of an optical fiber or rod-like waveguide in an immunoassay. Since light is confined inside an optical fiber by a series of internal reflections, an evanescent wave field exists along the entire surface of the fiber. Antibodies or antigens are attached to the fiber, and the fiber is then immersed in the sample to be tested. Fluorescence or other optical changes can be detected at an end of the fiber. Because some such devices can be immersed directly into a neat biological fluid, they are sometimes referred to as "biosensors". Whatever Hirschfeld's advantages, his system still requires cleaning or replacement of the biosensor after each use.
U.S. Pat. No. 4,810,658 (Shanks et al.) describe a waveguide which is placed in contact with an illuminated sample. Fluorescence from bound material produces an evanescent wave in the waveguide which exits the waveguide above the critical angle. Fluorescence from unbound material is refracted at the interface, and can therefore only exit the waveguide at an angle below the critical angle.
Systems such as those described by Schutt et al. and Shanks et al. rely upon optical apertures to limit the acceptance angle of the detector so that non-evanescent waves are excluded. The edge of the waveguide in these cases is an extended light source, as opposed to a point source. That is, light is emitted from regions that do not lie on the optical axis of the system. Under these conditions, no aperture can be designed that accepts all rays up to a given incidence angle, a, and rejects all others. Thus separation of evanescent and non-evanescent radiation will be less than ideal. Under most assay conditions, the evanescent signal is much weaker than the non-evanescent background, so good separation is essential. Furthermore, apertures must be properly aligned with respect to the waveguide and the detector in order to function well.
Another disadvantage of Shanks et al. is that the illuminating beam passes through the sample in order to reach the waveguide. This increases the interfering effects in the bulk solution of light scattering or absorbing substances on the evanescent wave signal.